Vol. 11(6), pp. 480-486, 11 February, 2016 DOI: 10.5897/AJAR2015.10748 Article Number: DE28DE157124 African Journal of Agricultural ISSN 1991-637X Copyright ©2016 Research Author(s) retain the copyright of this article http://www.academicjournals.org/AJAR
Full Length Research Paper
Evaluation of performance and chemical composition of Petroselinum crispum essential oil under different conditions of water deficit
Ivania Burin Borges1, Bruna Karen Cardoso1, Eloísa Schneider Silva1, Jéssica Souza de Oliveira1, Rafael Ferreira da Silva1, Cláudia Moraes de Rezende2, José Eduardo Gonçalves3, Ranulfo Piau Junior4, Silvia Graciele Hulse de Souza1 and Zilda Cristiani Gazim1*
1Programa de Pós-Graduação em Biotecnologia Aplicada à Agricultura. Universidade Paranaense, Umuarama, Praça Mascarenhas de Moraes, 4282, 87502-210 Umuarama, Paraná, Brasil. 2Centro de Tecnologia, Instituto de Quimica,Universidade Federal do Rio de Janeiro. Av. Athos da Silveira Ramos, 149, 21941-909, Rio de Janeiro, RJ, Brazil. 3Programa de Pós-graduação em Promoção da Saúde e em Tecnologias Limpas. Centro Universitário de Maringá, UniCESUMAR, Av. Guerdner no 1610, Jd. Aclimação, Maringá, PR, Brasil. 4Programa de Pós-Graduação em Ciência Animal. Universidade Paranaense, Umuarama, Praça Mascarenhas de Moraes, 4282, 87502-210 Umuarama, Paraná, Brasil.
Received 18 December, 2015; Accepted 19 January, 2016
This experiment aimed to assess the development, yield and chemical composition of Petroselinum crispum essential oil (EO), popularly known as parsley under different levels of water stress deficit: 30 to 40% (moderate stress), 50 to 60% (severe stress) and control 0 to 10%. The plants were kept in a greenhouse to complete the cycle, harvested and measured for their biomass (g), development (cm), yield (%) and chemical composition of EO of aerial parts and root. The EO was extracted by hydrodistillation and the chemical analysis done by gas chromatography/mass spectrometry (GC/MS). In the control and moderate stress, greater development of the aerial part occurred without showing difference in the root development. A yield increase of EO extracted from the roots was verified when it was submitted to moderate stress, but there was no difference in the aerial part yield. The EO chemical composition was influenced by the different conditions of water deficit, producing 100% of apiole in moderate stress and control of aerial parts and roots. A different behavior was observed for severe stress, which presented three compounds (89.98% apiole, 6.53% β-sesquiphellandrene and 3.49% myristicin) in aerial parts, but four compounds were found for the root (88.40% apiole, 5.83% β- sesquiphellandrene, 3.61% myristicin and 2.16% elemicin). These results indicated that under greater water deficit, the plant produced other compounds besides apiole, probably as a defense mechanism. The results showed that there was influence of the water stress on the plant development as well as on the essential oil yield and chemical composition.
Key words: Apiole, gas chromatography/mass spectrometry (GC/MS), essential oil, water stress, parsley.
INTRODUCTION
The species Petroselinum crispum (Mill.) belongs to the is an erect, evergreen herb with strong aromatic family Apiaceae, popularly known as parsley. This plant compound leaves, and inflorescences in the shape of Borges et al. 481
Table 1. Analysis of pH, macro and micronutrients of soil utilized in parsley cultivation.
pH and Macronutrients pH Al 3+ H+ + Al3+ Ca2+ Mg2+ K+ SB CTC P C -3 -3 -3 -3 3 3 -3 -3 (CaCl2) (cmoldm ) (cmoldm ) (cmol dm ) (cmol dm ) (cmol dm ) (cmol dm ) (mg dm ) (mg dm ) 6.91 0 1.89 13.25 0.21 13.46 15.35 188.0 23.38
Micronutrients V Ca Mg K Ca/Mg Ca/K Mg/K - - 87.68 57.02 21.94 1.34 1.94 42.66 21.94 - -
Ca, Mg, Al: extracted withKCl 1 mol L -1; P, K: extracted with Mehlic 1; H + Al: SMP method; C: Walkley & balcknethod; SB: Sum of Bases. Source: Laboratório Solo Fértil (Umuarama-PR).
terminal umbels over the leaves, with small yellow- 2011). greenish flowers. The fruits are achenes, and the plants Secondary metabolites are involved in different defense propagate by seeds, reaching 15 to 30 cm of height mechanisms under abiotic pressures, and water deficit is (Lorenzi and Matos, 2008; Vora et al., 2009). Parsley among the main causes of yield loss, but presents direct stands out because it is one of the most consumed herbs correlation with the concentration of secondary in the world. It can be utilized either in the food industry metabolites. There are reports in the literature that water or as medicinal plant. It can be used fresh or dehydrated, stress usually induces the increase in the concentration and in that case, the most consumed parts are leaves, of some terpenoids (Morais, 2009). Studies evaluating the petioles and seeds. The plants develop mostly on sandy influence of water stress on the production of P. crispum clay soils with high content of organic matter, good essential oil showed that under deficit conditions of 45 to fertility and pH between 5.8 and 6.8 (Heredia et al., 60%, the concentration of essential oil (EO) was higher 2003). The essential oil (EO) utilized to aromatize foods (44%) than fresh leaves (Petropoulos et al., 2008). is extracted from seeds, roots and leaves. The chemical High concentrations of secondary metabolites resulted composition of this oil consists of pinene, myrcene, in more tolerant plants, but the increase in secondary phellandrene, cymene, methatriene, elemene, myristicin metabolite production represents a high energetic and apiole (Petropoulos et al., 2008). demand from the plant which consequently reduces its Because of the worldwide demand for raw materials, growth, production, biomass and reproduction (Eulgem et essential oils have had an important role in the food, al., 1999). This metabolite increase, usually under deficit drug, cosmetic and perfume industries. They can situations, made plants evolve due to their induced substitute or act in synergism with chemical compounds, defense system. Thus, in order to evaluate the influence mainly the synthetic ones, since they have antimicrobial of water stress on parsley development, this study aimed potential (Farag et al., 1989; Kamel, 2000; Tzakou et al., to measure the development, yield and chemical 2001), besides having immomodulating function, composition of EO of P. crispum under different antioxidant and food preservative properties (Farag et al., conditions of water deficit. 1989). During their life cycle, plants are exposed to adverse conditions; among them are the different water stresses. MATERIALS AND METHODS Water deficit is one of the limiting factors of vegetal growth and development, since water works as a reagent Soil preparation in most metabolic reactions (Krieg, 1993). Several processes are altered in the plant due to water deficit The utilized soil presents sandy superficial texture with low contents such as gas exchange, carbohydrate metabolism, amino of clay and organic matter, and is classified as distroferric red acids and other organic compounds (Pagter et al., 2005). latosol (Fidalski et al., 2013). Thus, it was necessary to prepare the Therefore, to survive under such environmental soil since parsley needs organic matter to develop itself (Heredia et al., 2003). The soil was prepared using a mixture containing 65% of conditions, plants have developed a complex genetic- manure and 35% of soil with 710 µm granulometry. Later, it was molecular and cellular signaling network that makes them sent for analysis to Laboratório Solo Fértil (CRQ-03751 and CRF- respond through different metabolic pathways (Guo et al., PR 015963/0) located in the city of Umuarama, PR, Brazil (Table 1).
*Corresponding author. E-mail: [email protected].
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 482 Afr. J. Agric. Res.
Table 2. Influence of water deficit treatments on the development of aerial parts and roots of P. crispum. Control (0 to 10%), Moderate Stress (30 to 40%), Severe Stress (50 to 60%).
Control Moderate stress Severe stress Development (0-10%) (30-40%) (50-60%) Aerial part (cm) 20.0 ± 0.30a 18.31 ± 0.25b 15.33 ± 0.05c Aerial part (g) 144.54 ± 6.54a 128.33 ± 9.5a 90.36 ± 8.16b Roots (cm) 19.71 ± 0.084a 16.67 ± 0.17a 17.89 ± 0.17a Roots (g) 145.41 ± 9.32a 137.00 ± 9.00a 109.34 ± 5.85a
Different small letters on the same line indicate significant difference (p≤.0.05).
Obtaining plant vegetal m × 0.25 mm × 0.25 µm). The injector temperature was 250°C in “Split” mode 2.1:1, and the carrier gas was helium with a flow of 4.8 In November, 21-day-old seedlings of Portuguese P. crispum ml/min. The temperatures of the transfer line, ion source and cultivar, approximate size of 4.0 cm, were collected from their quadrupole were 320, 230 and 150°C, respectively. The column seedbed and transferred to 2 L vases. The experiment had random temperature was programmed for initial temperature of 40°C, design, was carried out in a greenhouse located in Paranaense heating rate of 6°C/min, and final temperature of 300°C (1 min), University - UNIPAR, in the city of Umuarama, northwestern region total analysis time of 44.33 min. The detection system was MS in of Paraná Sate, with coordinates S23° 46.225’ and WO 53° 16.730’ “Scan” mode, in the mass/load ratio range (m/z) of 50 to 550, with and altitude of 391 m. A voucher specimen was deposited at the "Solvent Delay" of 3.5 min (Petropoulos et al., 2008). The injections UNIPAR Herbarium (code number 192). After a month, the plants were done in triplicate for each irrigation pattern. were submitted to two treatments of water deficit in which irrigation was applied at soil water potentials of 30 to 40% (moderate stress) and of 50 to 60% (severe stress), whereas the control was 0 to 10% Statistical analysis (control). These percentages represent 0% of soil in field capacity and 100% of dry soil (Petropoulos et al., 2008). 40 vases were Data descriptive analysis was done and the data were expressed utilized for each treatment. The plants were kept in vases until by determining the averages and standard error through analysis of reaching flowering, when they were harvested. The length (cm) and variance (ANOVA) and Tukey’s test at 5% significance utilizing fresh mass (g) of their roots and aerial parts were measured. Miinitab®17 software.
Extraction of essential oil (EO) RESULTS AND DISCUSSION
The fresh material from aerial part and roots was submitted to hydrodistillation (Stanković et al., 2005; Petropoulos et al., 2008) for This study evaluated the influence of two different three hours, and the extractions were done in triplicate for each treatments of water deficit in the development of aerial treatment. The oil was removed from the device with hexane (PA) parts and roots (Table 2), and it was observed that and filtered with anhydrous Na2SO4, stored in amber flasks under different water stress conditions of 30 to 40% (moderate refrigeration and open until hexane had evaporated completely. After total evaporation of the solvent, each extract was weighed to stress) and 50 to 60% (severe stress) significantly calculate oil yield (%). influenced the development of aerial parts, but did not change the development of roots. In the aerial parts, moderate stress and control presented greater masses Physical and chemical indexes of essential oil obtained by (128.33 and 144.54 g, respectively) but did not differ hydro distillation between them when compared to severe stress that
The refraction and relative density indexes of P. crispum essential presented smaller mass (90.36 g). Plants with less oil were determined. The tests were carried out in the Laboratory of irrigation (severe stress) had smaller biomass Natural Product Chemistry of UNIPAR, Paranaense University, development, probably due to the low water levels that Campus of Umuarama, Brazil. The refraction index was determined caused stomata closing, decreasing photosynthesis rate by a ABBE-type refractometer, model RL3, PZO Warszawa, at and consequently, plant biomass (Tardieu et al., 1992). 20°C (FARMACOPÉIA BRASILEIRA, 1988). A substance relative There was no significant difference for the root length density is its mass ratio by the same water volume mass, both at 20°C according to the technique described in the Brazilian (cm) and mass (g) in the different treatments (Table 2). Pharmacopeia (FARMACOPÉIA BRASILEIRA, 1988). Cultivation in vases with greater density may produce smaller root biomass and length due to the smaller space for the plant growth (Marotti et al., 1994). Determining chemical compounds of essential oil through Studies carried out by Petropoulos et al. (2008) GC/MS evaluated the development of biomass (g) of aerial parts
The GC-MS analyses were performed utilizing an Agilent GC, 6890 and roots of parsley submitted to water deficit levels of (0 model, and for MS, 5973 Network Mass Selective Detector. The to 10%) control; (30 to 45%) level 1 and (45 to 60%) level column had 5% phenyl methylsiloxane (DB-5) and its size was (30 2, resulting in greater biomass of aerial parts (62.3 g) in Borges et al. 483
Table 3. Influence of water deficit treatments on the chemical and physical indexes of P.crispum EO, Control (0 to 10%), Moderate Stress (30 to 40%), Severe Stress (50 to 60%).
Chemical and physical indexes Refraction Index Relative density P. crispum essential oil 20 20 nD d 20 Aerial part Control (0-10%) 1.5305 ± 0.00a 1.27 ± 0.35a Moderate Stress (30-40%) 1.5310 ± 0.00a 1.65 ± 0.01a Severe Stress (50-60%) 1.5312 ± 0.00a 1.45 ± 0.42a
Root Control (0-10%) 1.5285 ± 0.00 a 1.27 ± 0.00a Moderate Stress (30-40%) 1.5250 ± 0.00 b 1.27 ± 0.00 a Severe Stress (50-60%) 1.5245 ± 0.00 c 1.15 ± 0.01a
Different small letters on the same column indicate significant difference (p≤0.05).
the control when compared to level 1 (32.8 g) and level 2 The results found for relative density and refraction (29.6 g). These results corroborate others found in this index are in accordance with the report by Stanković et experiment in which lower irrigation levels induced the al. (2005) who analyzed parsley oil from the region of decrease of plant biomass and resulted in (144.41 g) for Belgrade in Serbia, and found density ranging from 1.039 control, in (128.33 g) for moderate stress and in (90.36 g) to 1.061 g ml-1, and refraction index varying from 1.52214 for severe stress. For the roots, Petropoulos et al. (2008) to 1.5273. found biomass values (17.8, 11.3 and 7.3 g) inversely Regarding water stress in the yield of EO from aerial proportional to the increasing order of water deficit. parts and roots of P. crispum (Figure 1), the yield (%) of These values are different from the ones found in this EO from aerial parts did not present significant difference experiment in which there was inverse relation but not between the evaluated water deficit treatments; however, significant difference of the root biomass under the EO from roots showed significant difference between the different conditions of water stress (145.41, 137.00 and three water deficit parameters, as shown in Figure 1. 109.34 g). There is a greater yield of EO 0.14% for moderate stress Bettaieb et al. (2009) analyzed the influence of water and 0.01% for severe stress when compared to (0.05%) deficit levels that were severe (75%), moderate (50%) for control. These results are in accordance to the ones and control (0%) in Salvia officinalis plant, evaluating the obtained by Bettaieb et al. (2009) in Salvia officinalis EO. parameters of aerial part biomass (g) and length (cm), However, Petropoulos et al. (2008) also assessed the and found a reduction of biomass (4.96%) and length yield of EO from aerial parts and root of parsley but did (14.15 cm) under severe water deficit condition (12.63%, not find a difference between the evaluated water deficit 19.78 cm) for moderate water deficit, and (19.53%, 26.50 treatments. cm) for control, respectively. These results are in The difference of water deficit levels in the yield of accordance with the present experiment, making it Pimpinela anisum EO, also belonging to the family evident that the greater the water deficit the slower the Apiaceae, was studied by Zehtab et al. (2001) who plant development. observed greater oil yields when the plant was submitted This experiment evaluated the influence of water deficit to a water stress standard below 20%. Farahani et al. in the physical and chemical indexes of parsley, (2009) assessed the influence of four water deficit levels measuring the density (g ml-1) and the refraction index, as (20, 40, 60 and 80%) on Melissa officinalis plant, and shown in Table 3. The results showed that there was no found the greatest EO yield at 40% water stress. The significant difference for the density of EO from aerial cited experiments corroborate the results found in this parts and roots under the evaluated water stress study in which there was a greater yield of EO from the conditions. For the refraction index of EO, there was no roots when the plant was submitted to water deficit of 30 difference among the aerial parts; however, there was a to 40%. According to Morais (2009), alterations of the significant difference of this parameter of EO in the two essential oil biosynthesis may function as an adaptive evaluated treatments and control. The refraction index response to water stress, relating some physiological has direct correlation with the density, but this response to environmental variations. relationship was not observed in the results presented in Through the chemical analysis by GC/MS, 10 Table 3. compounds were found in P. crispum EO from the aerial 484 Afr. J. Agric. Res.
Figure 1. Influence of water deficit treatments in the yield of essential oil from aerial parts and roots of P. (a) crispum. Control (0 to 10%), Moderate Stress (30 to 40%), Severe Stress (50 to 60%) in the chemical composition of EO from aerial parts and roots of P. crispum.
Figure 2. Chromatograms obtained by GC/MS of essential oil of P. crispum from the aerial parts and roots submitted to two (a) cis-Muurolol-5-en-4- water deficit treatments. Control (0 to 10%), moderate stress (30 to 40%), severe stress (50 to 60%).
ß-ol
parts and roots that are listed in elution order in Table 4 moderate stress made evident the presence of 100% of and Figure 2. The water deficit of the control and apiole in EO from aerial parts and roots. Borges et al. 485
Table 4. Influence of water deficit treatments on the chemical composition of EO from aerial parts and roots of P. crispum. Control (0-10%), Moderate Stress (30-40%), (a) Severe Stress (50-60%).
Aerial Part Root Identification Peak CompoundsA aRI lit. bRI calc. Control Moderate stress Severe stress Control Moderate stress Severe methods (0-10%) (30-40%) (50-60%) (0-10%) (30-40%) stress (50-60%) 1 β- phellandrene 1025 1000 t t t t t t a, b, c 2 Camphor 1141 1100 t t t t t t a, b, c 3 β -elemene 1389 1326 t t t t t t a, b, c 4 β- Caryophyllene 1419 1446 t t t t t t a, b, c 5 β-bisabolene 1484 1480 t t t t t t a, b, c 6 Myristicin 1517 1501 t t 3.49± 0.02 t t 3.61 ± 0.02 a, b, c 7 β -sesquiphellandrene 1521 1505 t t 6.53 ± 0.03 t t 5.83 ± 0.04 a, b, c 8 γ-bisabolene (E) 1529 1509 t t t t t t a, b, c 9 Elemicin 1555 1523 t t t t t 2.16 ± 0.01 a, b, c 10 Apiole 1677 1643 100 ± 0.00 100 ± 0.00 89.98±0.05 100 ± 0.00 100 ± 0.00 88.40 ± 0.09 a, b, c
The values of the areas (%) obtained by GC/MS, were calculated from the averages ± standard error from three independent replications. Identification Methods: aRIcalc.-Calculated b retention index utilizing n-alcans C7 – C25 in column DB-5 (phenylmethylsiloxane 5%). RI lit.-Relative retention index found in literature of the capillary column DB5 and comparison of Retention Indexes and/or Mass Spectra with literature (ADAMS, 2007). cMS-Identification based on the comparison of mass spectra with Wiley 275 libraries t-traces. ACompounds listed according to the elution order by column DB-5 ((phenylmethylsiloxane %).
In severe stress, EO from the aerial parts aminoacid in a reaction catalyzed by 12.97 and 21.00%) only in the roots. The results contained apiole (89.98%), β-sesquiphellandrene phenylalanine ammonium liase (PAL).The activity from this experiment and from literature (6.53%) and myristicin (3.49%) whereas EO from of this enzyme in plants is increased as a (Petropoulos et al., 2008) showed that the the roots had apiole (88.40%), β- consequence of biotic and abiotic stresses and chemical composition of parsley EO was sesquiphellandrene (5.83%), myristicin (3.61%) then a lot of phenylpropanoids are produced, influenced directly by the different levels of water and elemicin (2.16%), respectively. These results justifying the increase of myristicin and elemicin deficit (Table 4 and Figure 1). In our experiment, indicated that under greater water deficit (severe when the plant was submitted to the water deficit the main compound in all treatments of water stress), the plant responded by decreasing the (Dixon and Paiva, 1995). deficit was apiole. Petropoulos et al. (2008) found content of apiole and increasing the amount of Petropoulos et al. (2008) also evaluated the myristicin and β-phellandrene as the main myristicin, β-sesquiphelandrene and elemicin, influence of three levels of water deficit: (0 to compounds. This difference in the chemical beside apiole, probably as a defense mechanism 10%), (30 to 45%) and (45 to 60%) in the composition probably occurred due to the cultivar, (Morais and Castanha, 2012). Similar results were chemical composition of parsley EO from aerial utilized substrate, geographical location, and found by Ekren et al. (2012) in which there was an parts and roots cultivated in Greece. Ten main weather since they are factors that interfere in the increase in the number of Ocimum basilicum oil compounds were found: myristicin (28.63, 33.43 chemical composition of EO (Morais and compounds as there was an increase in water and 33.35%) in the aerial parts and (13.55, 16.05 Castanha, 2012). deficit. This increase of compounds may be and 7.7%) in the roots, β phellandrene (25.07, These results are promising when the metabolically explained because the main 23.63 and 21.97%) in the aerial parts and (20.95, experimental objective is to isolate main compounds of parsley oil are made of 14.37 and 19.8%) in the roots, and apiole (2.91, compounds for commercial purposes because phenylproanoids that are derived from trans 1.90 and 6.35%) in the aerial parts and (23.59, studies done on P. crispum EO showed that cinamic acid which is formed by phenylalanine 33.92 and 25.70%) in the roots; β-pinene (16.43, phenylpropanoids like apiole and myristicin have 7
486 Afr. J. Agric. Res.
antioxidant properties; myristicin can also be utilized as a Farahani HA, Valadabadi SA, Daneshian J, Khalvati MA chemopreventive (Reyes-Munguía et al., 2012) and (2009).Evaluation changing of essential oil of balm (Melissa officinalis L.) under water deficit stress conditions. J. Med. Plant Res. 3(5):329- apiole can be used in several biological activities, mainly 333. antifungal ones (Farag et al., 1989). FARMACOPÉIA BRASILEIRA (1988). 4ª Ed. São Paulo: Atheneu. Fidalski J, Tormena CA, Alves SJ, Auler PAM (2013). Influence of sand fractions on water retention and availability in caiuá and Paranavaí sandstone formations. Rev. Bras. Cienc. Solo. 37(3):613-621. Conclusion Guo R, Yu F, Gao Z, An H, Cao X, Guo X (2011). GhWRKY3 a novel cotton (Gossypium hirsutum L.)WRKY gene, is involved in diverse This experiment made evident that when the plant was stress responses. Mol. Bio. Rep. 38(1):49-58. submitted to a greater water stress condition (severe Heredia ZNA, Vieira MC, Weisman LAL (2003). Bunching onion and parsley yield and gross income in mono-cropping and inter-cropping stress) there was a decrease of the aerial part system. Hortic. Bras. 21(3):574-577. development without interfering in the root development. Kamel C (2000). A novel look at a classic approach of plant extracts Moderate stress provided greater yield (%) of EO from (special number) Feed Mix. The Int. J. Feed Nutr. Technol. 9(6):19- roots without changing the yield of aerial parts. The 24. Krieg DR (1993). Stress tolerance mechanisms in above ground chemical composition of the EO showed apiole as the organs. Proceedings of the Workshop on Adaptation of Plants to Soil main compound in all evaluated treatments, 100% for the Stress.2ª Ed. Nebraska: Intsormil. pp. 65-79. control and moderate stress. However, for severe stress Lorenzi H, Matos FJA (2008). Plantas medicinais no Brasil - Nativas e (greater water deficit), myristicin, elemicin and β- exóticas.2ªEd. Nova Odessa: Plantarum 544p. Marotti M, Piccaglia R, Giovanelli E, Deans SG, Eaglesham E (1994). sesquiphellandrene were also found, besides apiole. Effects of planting time and mineral fertilization on peppermint These results are promising to obtain isolated (Mentha x piperita L.) essential oil composition and its biological compounds utilized to determine biological activities and activity. Flavour Fragr. J. 9(3):125-129. to be utilized in the food and pharmaceutical industries. Morais LAS, Castanha RF (2012). Chemical composition of sweet basil essential oil naturally submitted to Planococcus citri infestation. Hortic. Bras. 30(2):S2178-S2182. Morais LAS (2009). Influência dos fatores abióticos na composição Conflict of interests química dos óleos essenciais. Hort. Brás. 27: 4050-4063. Pagter M, Bragato G, Brix H (2005). Tolerance and physiological responses of Phragmites australis to water deficit. Aquat. Bot. The author(s) have not declared any conflict of interests. 81(4):285-299. Petropoulos SA, Daferera D, Polissiou MG, Passam HC (2008). The effect of water deficit stress on the growth, yield and composition of ACKNOWLEDGEMENTS essential oils of parsley. Sci. Hortic. 115(4):393-397. Reyes-Munguía A, Zavala-Cuevas D, Alonso-Martínez A (2012). Perejil (Petroselinum crispum):compuestos químicos y aplicaciones. The authors thank CAPES, Paranaense University Tlatemoani: Revista Académica de Investigación. 11:18. (UNIPAR), the Institute of Chemistry of the Federal Stanković M, Nikolić N, Stanojević L, Petrović S, Cakić M (2005). University of Rio de Janeiro (IQ-UFRJ) and the University Hydrodistillation kinetics and essential oil composition from fermented parsley seeds. CI&CEQ.11:25-29. Center of Maringá (CESUMAR) for the support. Tardieu F, Bruckler L, Lafolie F (1992). 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